[FeFe]-hydrogenase abundance and diversity along a vertical redox gradient in Great Salt Lake, USA.

The use of [FeFe]-hydrogenase enzymes for the biotechnological production of H2 or other reduced products has been limited by their sensitivity to oxygen (O2). Here, we apply a PCR-directed approach to determine the distribution, abundance, and diversity of hydA gene fragments along co-varying salinity and O2 gradients in a vertical water column of Great Salt Lake (GSL), UT. The distribution of hydA was constrained to water column transects that had high salt and relatively low O2 concentrations. Recovered HydA deduced amino acid sequences were enriched in hydrophilic amino acids relative to HydA from less saline environments. In addition, they harbored interesting variations in the amino acid environment of the complex H-cluster metalloenzyme active site and putative gas transfer channels that may be important for both H2 transfer and O2 susceptibility. A phylogenetic framework was created to infer the accessory cluster composition and quaternary structure of recovered HydA protein sequences based on phylogenetic relationships and the gene contexts of known complete HydA sequences. Numerous recovered HydA are predicted to harbor multiple N- and C-terminal accessory iron-sulfur cluster binding domains and are likely to exist as multisubunit complexes. This study indicates an important role for [FeFe]-hydrogenases in the functioning of the GSL ecosystem and provides new target genes and variants for use in identifying O2 tolerant enzymes for biotechnological applications.

Contrasting patterns of community assembly in the stratified water column of Great Salt Lake, Utah.

Phylogenetic examinations of communities sampled along geochemical gradients provide a framework for inferring the relative importance of niche-based ecological interactions (competition, environmental filtering) and neutral-based evolutionary interactions in structuring biodiversity. Great Salt Lake (GSL) in Utah exhibits strong spatial gradients due to both seasonal variation in freshwater input into the watershed and restricted fluid flow within North America's largest saline terminal lake ecosystem. Here, we examine the phylogenetic structure and composition of archaeal, bacterial, and eukaryal small subunit (SSU) rRNA genes sampled along a stratified water column (DWR3) in the south arm of GSL in order to infer the underlying mechanism of community assembly. Communities sampled from the DWR3 epilimnion were phylogenetically clustered (i.e., coexistence of close relatives due to environmental filtering) whereas those sampled from the DWR3 hypolimnion were phylogenetically overdispersed (i.e., coexistence of distant relatives due to competitive interactions), with minimal evidence for a role for neutral processes in structuring any assemblage. The shift from phylogenetically clustered to overdispersed assemblages was associated with an increase in salinity and a decrease in dissolved O2 (DO) concentration. Likewise, the phylogenetic diversity and phylogenetic similarity of assemblages was strongly associated with salinity or DO gradients. Thus, salinity and/or DO appeared to influence the mechanism of community assembly as well as the phylogenetic diversity and composition of communities. It is proposed that the observed patterns in the phylogenetic composition and structure of DWR3 assemblages are attributable to the meromictic nature of GSL, which prevents significant mixing between the epilimnion and the hypolimnion. This leads to strong physicochemical gradients at the halocline, which are capable of supporting a greater diversity. However, concomitant shifts in nutrient availability (e.g., DO) at and below the halocline drive competitive interactions leading to hypolimnion assemblages with minimal niche overlap.

Alternative carbon sources for the production of plant cellular agriculture: a case study on acetate.

Plant cellular agriculture aims to disrupt the way plant derived products are produced. Plant cell cultures are typically grown with sucrose as the primary carbon and energy source, but alternative carbon sources may have advantages over sucrose including less strain on food systems, lower costs, and more sustainable sourcing. Here we review carbon and energy sources that may serve as alternatives to sucrose in the cultivation of plant cell cultures. We identified acetate as a promising candidate and took the first steps to evaluate its potential for use in growing tobacco plant cell cultures. When added to media containing sucrose, acetate concentrations above 8 mM completely inhibit growth. Lower concentrations of acetate (2-4 mM) can support an increase in dry weight without sucrose but do not provide enough energy for substantial growth. 13C labeling indicates that tobacco plant cell cultures can incorporate carbon from exogenous acetate into proteins and carbohydrates. Analysis of transcriptome data showed that genes encoding glyoxylate cycle enzymes are expressed at very low levels compared to genes from the TCA cycle and glycolysis. Adaptive laboratory evolution experiments were able to increase tobacco cell cultures tolerance to acetate, demonstrating the potential for this type of approach going forward. Overall, our results indicate that acetate can be metabolized by plant cell cultures and suggest that further adaptive laboratory evolution or strain engineering efforts may enable acetate to serve as a sole carbon and energy source for tobacco plant cell cultures. This assessment of acetate provides a framework for evaluating other carbon and energy sources for plant cell cultures, efforts that will help reduce the costs and environmental impact, and increase the commercial potential of plant cellular agriculture.

Genetic disruption of both Chlamydomonas reinhardtii [FeFe]-hydrogenases: Insight into the role of HYDA2 in H2 production.

Chlamydomonas reinhardtii (Chlamydomonas throughout) encodes two [FeFe]-hydrogenases, designated HYDA1 and HYDA2. While HYDA1 is considered the dominant hydrogenase, the role of HYDA2 is unclear. To study the individual functions of each hydrogenase and provide a platform for future bioengineering, we isolated the Chlamydomonas hydA1-1, hydA2-1 single mutants and the hydA1-1 hydA2-1 double mutant. A reverse genetic screen was used to identify a mutant with an insertion in HYDA2, followed by mutagenesis of the hydA2-1 strain coupled with a H(2) chemosensor phenotypic screen to isolate the hydA1-1 hydA2-1 mutant. Genetic crosses of the hydA1-1 hydA2-1 mutant to wild-type cells allowed us to also isolate the single hydA1-1 mutant. Fermentative, photosynthetic, and in vitro hydrogenase activities were assayed in each of the mutant genotypes. Surprisingly, analyses of the hydA1-1 and hydA2-1 single mutants, as well as the HYDA1 and HYDA2 rescued hydA1-1 hydA2-1 mutant demonstrated that both hydrogenases are able to catalyze H(2) production from either fermentative or photosynthetic pathways. The physiology of both mutant and complemented strains indicate that the contribution of HYDA2 to H(2) photoproduction is approximately 25% that of HYDA1, which corresponds to similarly low levels of in vitro hydrogenase activity measured in the hydA1-1 mutant. Interestingly, enhanced in vitro and fermentative H(2) production activities were observed in the hydA1-1 hydA2-1 strain complemented with HYDA1, while maximal H(2)-photoproduction rates did not exceed those of wild-type cells.

Evolutionary significance of an algal gene encoding an [FeFe]-hydrogenase with F-domain homology and hydrogenase activity in Chlorella variabilis NC64A.

[FeFe]-hydrogenases (HYDA) link the production of molecular H(2) to anaerobic metabolism in many green algae. Similar to Chlamydomonas reinhardtii, Chlorella variabilis NC64A (Trebouxiophyceae, Chlorophyta) exhibits [FeFe]-hydrogenase (HYDA) activity during anoxia. In contrast to C. reinhardtii and other chlorophycean algae, which contain hydrogenases with only the HYDA active site (H-cluster), C. variabilis NC64A is the only known green alga containing HYDA genes encoding accessory FeS cluster-binding domains (F-cluster). cDNA sequencing confirmed the presence of F-cluster HYDA1 mRNA transcripts, and identified deviations from the in silico splicing models. We show that HYDA activity in C. variabilis NC64A is coupled to anoxic photosynthetic electron transport (PSII linked, as well as PSII-independent) and dark fermentation. We also show that the in vivo H(2)-photoproduction activity observed is as O(2) sensitive as in C. reinhardtii. The two C. variabilis NC64A HYDA sequences are similar to homologs found in more deeply branching bacteria (Thermotogales), diatoms, and heterotrophic flagellates, suggesting that an F-cluster HYDA is the ancestral enzyme in algae. Phylogenetic analysis indicates that the algal HYDA H-cluster domains are monophyletic, suggesting that they share a common origin, and evolved from a single ancestral F-cluster HYDA. Furthermore, phylogenetic reconstruction indicates that the multiple algal HYDA paralogs are the result of gene duplication events that occurred independently within each algal lineage. Collectively, comparative genomic, physiological, and phylogenetic analyses of the C. variabilis NC64A hydrogenase has provided new insights into the molecular evolution and diversity of algal [FeFe]-hydrogenases.

Increased lipid accumulation in the Chlamydomonas reinhardtii sta7-10 starchless isoamylase mutant and increased carbohydrate synthesis in complemented strains.

The accumulation of bioenergy carriers was assessed in two starchless mutants of Chlamydomonas reinhardtii (the sta6 [ADP-glucose pyrophosphorylase] and sta7-10 [isoamylase] mutants), a control strain (CC124), and two complemented strains of the sta7-10 mutant. The results indicate that the genetic blockage of starch synthesis in the sta6 and sta7-10 mutants increases the accumulation of lipids on a cellular basis during nitrogen deprivation relative to that in the CC124 control as determined by conversion to fatty acid methyl esters. However, this increased level of lipid accumulation is energetically insufficient to completely offset the loss of cellular starch that is synthesized by CC124 during nitrogen deprivation. We therefore investigated acetate utilization and O(2) evolution to obtain further insights into the physiological adjustments utilized by the two starchless mutants in the absence of starch synthesis. The results demonstrate that both starchless mutants metabolize less acetate and have more severely attenuated levels of photosynthetic O(2) evolution than CC124, indicating that a decrease in overall anabolic processes is a significant physiological response in the starchless mutants during nitrogen deprivation. Interestingly, two independent sta7-10:STA7 complemented strains exhibited significantly greater quantities of cellular starch and lipid than CC124 during acclimation to nitrogen deprivation. Moreover, the complemented strains synthesized significant quantities of starch even when cultured in nutrient-replete medium.

Application of gene-shuffling for the rapid generation of novel [FeFe]-hydrogenase libraries.

A gene-shuffling technique was identified, optimized and used to generate diverse libraries of recombinant [FeFe]-hydrogenases. Six native [FeFe]-hydrogenase genes from species of Clostridia were first cloned and separately expressed in Escherichia coli concomitantly with the assembly proteins required for [FeFe]-hydrogenase maturation. All enzymes, with the exception of C. thermocellum HydA, exhibited significant activity when expressed. Single-stranded DNA fragments from genes encoding the two most active [FeFe]-hydrogenases were used to optimize a gene-shuffling protocol and generate recombinant enzyme libraries. Random sampling demonstrates that several shuffled products are active. This represents the first successful application of gene-shuffling using hydrogenases. Moreover, we demonstrate that a single set of [FeFe]-hydrogenase maturation proteins is sufficient for the heterologous assembly of the bioinorganic active site of several native and shuffled [FeFe]-hydrogenases.

Cyanophora paradoxa genome elucidates origin of photosynthesis in algae and plants.

The primary endosymbiotic origin of the plastid in eukaryotes more than 1 billion years ago led to the evolution of algae and plants. We analyzed draft genome and transcriptome data from the basally diverging alga Cyanophora paradoxa and provide evidence for a single origin of the primary plastid in the eukaryote supergroup Plantae. C. paradoxa retains ancestral features of starch biosynthesis, fermentation, and plastid protein translocation common to plants and algae but lacks typical eukaryotic light-harvesting complex proteins. Traces of an ancient link to parasites such as Chlamydiae were found in the genomes of C. paradoxa and other Plantae. Apparently, Chlamydia-like bacteria donated genes that allow export of photosynthate from the plastid and its polymerization into storage polysaccharide in the cytosol.

Insights into [FeFe]-hydrogenase structure, mechanism, and maturation.

Hydrogenases are metalloenzymes that are key to energy metabolism in a variety of microbial communities. Divided into three classes based on their metal content, the [Fe]-, [FeFe]-, and [NiFe]-hydrogenases are evolutionarily unrelated but share similar nonprotein ligand assemblies at their active site metal centers that are not observed elsewhere in biology. These nonprotein ligands are critical in tuning enzyme reactivity, and their synthesis and incorporation into the active site clusters require a number of specific maturation enzymes. The wealth of structural information on different classes and different states of hydrogenase enzymes, biosynthetic intermediates, and maturation enzymes has contributed significantly to understanding the biochemistry of hydrogen metabolism. This review highlights the unique structural features of hydrogenases and emphasizes the recent biochemical and structural work that has created a clearer picture of the [FeFe]-hydrogenase maturation pathway.

Phenotypic diversity of hydrogen production in chlorophycean algae reflects distinct anaerobic metabolisms.

Several species of green algae use [FeFe]-hydrogenases to oxidize and/or produce H(2) during anoxia. To further define unique aspects of algal hydrogenase activity, the well-studied anaerobic metabolisms of Chlamydomonas reinhardtii were compared with four strains of Chlamydomonas moewusii and a Lobochlamys culleus strain. In vivo and in vitro hydrogenase activity, starch accumulation/degradation, and anaerobic end product secretion were analyzed. The C. moewusii strains showed the most rapid induction of hydrogenase activity, congruent with high rates of starch catabolism, and anoxic metabolite accumulation. Intriguingly, we observed significant differences in morphology and hydrogenase activity in the C. moewusii strains examined, likely the result of long-term adaptation and/or genetic drift during culture maintenance. Of the C. moewusii strains examined, SAG 24.91 showed the highest in vitro hydrogenase activity. However, SAG 24.91 produced little H(2) under conditions of sulfur limitation, which is likely a consequence of its inability to utilize exogenous acetate. In L. culleus, hydrogenase activity was minimal unless pulsed light was used to induce significant H(2) photoproduction. Overall, our results demonstrate that unique anaerobic acclimation strategies have evolved in distinct green algae, resulting in differential levels of hydrogenase activity and species-specific patterns of NADH reoxidation during anoxia.